Metalloproteinase and encoding DNA therefor

ABSTRACT

A novel metalloproteinase, DNA encoding therefor, a plasmid carrying said DNA sequence and a host cell harbouring said plasmid, and monoclonal antibodies peculiarly recognizing said protein. 
     Useful in applications pertaining to diagnosis of the presence of tumour cells, the degree of cancer malignancy, and other medical and physiological fields.

This application is a continuation-in-part of PCT internationalapplication No. PCT/JP94/02009 which has an international filing date ofNov. 30, 1994 which designated the United States, the entire contents ofwhich are hereby incorporated by reference.

TECHNICAL FIELD

The present invention relates to a novel metalloproteinase useful inapplications such as diagnosis of the presence of tumour cells,diagnosis of the degree of tumour malignancy, or other medical orphysiological fields.

More specifically, the present invention relates to one type ofmetalloproteinase expressed specifically in human tumour cells and a DNAsequence encoding therefor; a plasmid having a nucleotide sequence whichcontains said DNA sequence; a host cell harbouring said plasmid; amethod for manufacturing said protein using said host cell; a probewhich hybridizes with the aforesaid DNA sequence; a method for detectingDNA or RNA containing the aforesaid sequence using said probe; andmonoclonal antibodies which bind specifically to the aforesaid protein.

BACKGROUND

A group of enzymes with different substrate specificity and referred toin general as matrix metalloproteinases (hereinafter referred to as“MMPs”) contributes to degradation of the extracellular matrixcomprising such complex components as collagen, proteoglycan, elastin,fibronectin, and laminin.

Previously reported MMPs include interstitial collagenase (MMP-1), 72kDa gelatinase (also known as type IV collagenase or gelatinase A;MMP-2), 92 kDa gelatinase (also known as type IV collagenase orgelatinase B; MMP-9), stromelysin-1 (MMP-3), matrilysin (MMP-7),neutrophil collagenase (MMP-8), stromelysin-2 (MMP10) and stromelysin-3(MMP-11).

These MMPs are a family of enzymes whose primary structure has beenreported previously. With the exception of MMP-7, the primary structureamong the family of reported MMPs comprises essentially an N-terminalpropeptide domain, a Zn⁺ binding catalytic domain and a C-terminalhemopexin-like domain. In MMP-7 there is no hemopexin-like domain. MMP-2and MMP-9 contain an additional gelatin-binding domain. In addition, aproline-rich domain highly homologous to a type V collagen α2 chain isinserted in MMP9 between the Zn⁺ binding catalytic domain and theC-terminal hemopexin-like domain.

In highly metastatic tumour cells, there are reports of conspicuousexpression of type IV collagenase (MMP-2, MMP-9) which mainly degradetype IV collagen (Cancer Res., 46:1-7, 1986; Biochem. Biophys. Res.Commun., 154:832-838, 1988; Cancer, 71:1368-1383, 1993). Likewise, ithas been reported MMP-3 act as an activator of proMMP-9 (J. Biol. Chem.,267:3581-3584, 1992).

The degree of matrix metalloproteinase expression serves as an index todiagnosing the degree of cancer malignancy.

DISCLOSURE OF THE INVENTION

The present inventors discovered a novel matrix metalloproteinase(hereinafter referred to as “MT-MMP”) and performed a structuralanalysis thereof.

As described hereafter, the present invention offers a novelmetalloproteinase protein, DNA having a nucleotide sequence whichencodes said protein, a plasmid having said DNA nucleotide sequence, ahost cell harbouring said plasmid and monoclonal antibodies whichspecifically recognize the aforesaid metalloproteinase protein.

1. A native membrane-type matrix-metalloproteinase characterized by acontinuous sequence of hydrophobic amino acids peculiar tomembrane-binding proteins from amino acid number 533 to 562 in the Cterminus domain shown in SEQ ID NO:1.

2. A native membrane-type matrix-metalloproteinase according to claim 1,characterized by the amino acid sequence from amino acid number 160 to173, 320 to 333 and from 498 to 512 shown in SEQ ID NO:1.

3. A native membrane-type matrix-metalloproteinase according to claim 1,characterized by the amino acid sequence from amino acid number 1 to173, 320 to 333, 498 to 512 and 563 to 582 shown in SEQ ID NO:1.

4. A DNA having the nucleotide sequence shown in SEQ ID NO:2 whichcorresponds to the amino acid sequence of a membrane-typematrix-metalloproteinase according to claim 1, 2 or 3.

5. A plasmid containing a DNA having the nucleotide sequence accordingto claim 4 and expressing a membrane-type matrix-metalloproteinaseaccording to claim 1, 2 or 3.

6. A host cell harbouring a plasmid containing a DNA having thenucleotide sequence according to claim 4, and expressing a membrane-typematrix-metalloproteinase according to claim 1, 2 or 3.

7. Monoclonal antibodies which peculiarly recognize a membrane-typematrix-metalloproteinase according to claim 1, 2 or 3.

8. A protein having the amino acid sequence shown in SEQ ID NO:1.

9. A DNA having the nucleotide sequence shown in SEQ ID NO:2 whichencodes a protein having the amino acid sequence shown in SEQ ID NO:1.

10. A plasmid containing a DNA having the nucleotide sequence shown inSEQ ID NO:2, and expressing the protein shown in SEQ ID NO:1.

11. A host cell harbouring a plasmid containing a DNA having thenucleotide sequence shown in SEQ ID NO:2, and expressing the proteinshown in SEQ ID NO:1.

12. Monoclonal antibodies which peculiarly recognize a protein havingthe amino acid sequence shown in SEQ ID NO: 1.

The present invention is described in detail hereafter.

Using highly conserved sequences (SEQ ID NOS:3 and 4) selected fromamino acid sequences of the known matrix metalloproteinase (MMP) family,the present inventors designed and synthesized an oligonucleotide primerhaving the sequences denoted by SEQ ID NOS:5 and 6. A PCR was carriedout using said oligonucleotide primer and a human placental cDNAlibrary, the PCR products obtained were sequenced, and a 390 bp DNAfragment having a sequence non-homologous to known MMP was obtained.Using this 390 bp DNA fragment as a probe, the human placenta cDNAlibrary was screened, and a cDNA in the positive phage clone obtainedwas sequenced. The nucleotide sequence is that denoted by SEQ ID NO:2. Asequence identical to the nucleotide sequence in SEQ ID NO:2 did notexist in the Genbank/EMBL DNA database, and DNA having this nucleotidesequence was ascertained to be completely novel.

The nucleotide sequence of the aforesaid cloned cDNA in SEQ ID NO:2 hada 3′ non-coding sequence and open reading frame that potentially encode582 amino acid. An initiation codon was located at nucleotide number112, and a stop codon was present at nucleotide number 1858. It wasdetermined that this open reading frame encoded the 582 amino acidsequence in SEQ ID NO:1, that a deduced signal sequence continued afterthe initiation codon, and that a hydrophobic domain (SEQ ID NO:7)specific to a membrane-binding protein of 20 or more linked hydrophobicamino acids was present from C-terminal amino acid number 533 to 562.

When homology between the amino acid sequence of MT-MMP and that of theknown MMP family was analyzed, MT-MMP had high homology to the known MMPfamily, as shown in FIG. 2. The sequences best conserved in MT-MMP wereactive site sequences, as well as sequences proximal to processing sitebetween precursor and mature substance conserved in the MMP family. Thefact that MT-MMP has the structural characteristics of amembrane-binding protein, and the presence in MT-MMP of a sequence oflinked hydrophobic amino acids (shown in Sequence Sheet sequence number7) not found in the rest of the MMP family, strongly suggested thatMT-MMP, unlike other MMP family, is a membrane-binding MMP.

When MT-MMP expression in various human tissues was studied by NorthernBlot analysis with various tissue-derived Poly(A)RNA, high expressionwas seen in the placenta, lung and kidney (see FIG. 3). Likewise,results from Northern Blot analysis performed with RNA extracted fromnormal and tumour areas of human lung squamous cell carcinoma showedthat MT-MMP is expressed peculiarly at tumour sites (see FIG. 4).

Finally, immunoprecipitation and immunostain experiments usinganti-MT-MMP monoclonal antibodies showed that the MT-MMP pertaining tothe present invention is expressed on a cell membrane without secretionof a gene product, and MMP-2 activation induced by the expression ofMT-MMP was observed in the cells transfected with MT-MMP gene (Nature,370:61-65, 1994).

Due to the achievements of the above-discussed research by the presentinventors, the present invention offers a novel matrix metalloproteinaseprotein having the amino acid sequence in SEQ ID NO:1.

In addition, the present invention offers DNA having the nucleotidesequence in SEQ ID NO:2, which encodes a protein having the amino acidsequence in SEQ ID NO:1; a plasmid containing and capable of expressingsaid DNA; and a host cell harbouring said plasmid. All host cells usedin general recombinant DNA technology can be used as the aforementionedhost cell, including prokaryotes such as E. coli and Bacilus subtilus;eukaryotes such as yeast, COS cells, CHO cells and 3T3 cells; and insectcells such as Sf21. Expression vectors corresponding to used host cellscan be used as the aforementioned plasmid.

Furthermore, the present invention offers mRNA transcribed from DNAhaving the nucleotide sequence in SEQ ID NO:2.

The present invention also offers a probe which hybridizes with theaforementioned DNA or RNA and specifically detects said DNA or RNA, andsaid probe may be one having any part of the nucleotide sequence in SEQID NO:2, provided said probe is labeled by a generally used radioactiveisotope or enzyme or the like, hybridizes specifically with said DNA orRNA in general blotting analysis and in situ hybridization, andaccomplishes detection.

Furthermore, the present invention offers monoclonal antibodies whichbind peculiarly with the MT-MMP pertaining to the present invention.

The monoclonal antibodies pertaining to the present invention can beprepared by a well-known method such as the method of Milstein et al.(Nature, 256:495-497, 1975) using human MT-MMP as an antigen. In thismethod, the antigen may be native human MT-MMP, recombinant humanMT-MMP, or a synthetic peptide having a partial amino acid sequence ofeither.

By means of the present invention, DNA having a nucleotide sequencewhich encodes a protein with the amino acid sequence of the novel MT-MMPpertaining to the present invention can be cloned, and such DNA and aprotein encoded by such DNA can be prepared by a genetic engineeringtechnique. Through the use of a cDNA clone of such a novel MT-MMP,techniques generally used in genetic engineering can be used to clonethe aforementioned nucleotide sequence into another vector or host.Based on the aforementioned cDNA nucleotide sequence, DNA appropriatelysuited to a probe may be designed and prepared. In addition, based onthe nucleotide sequence of the MT-MMP pertaining to the presentinvention, techniques generally used in genetic engineering can be usedto prepare a corresponding protein wherein appropriate mutation havebeen introduced into the MT-MMP amino acid sequence by substitution,deletion, insertion, displacement or addition of one or more aminoacids. All such aforementioned derivatives may also be included in thepresent invention, provided that common metalloproteinasecharacteristics are conserved; namely, sequences proximal to processingsite between precursor and mature substance, active site sequences anddomain structure, and provided that the MT-MMP characteristic of ahydrophobic domain of linked hydrophobic amino acids present near the Cterminus is conserved.

Use of the above-discussed various implementations of the presentinvention offers various technical means applicable to applicationspertaining to diagnostic agents or diagnostic methods used for diagnosisof the presence of tumour cells or for diagnosis of the degree of tumourmalignancy, as well as applications in other medical or physiologicalfields.

The present invention is described in detail hereafter by means ofWorking Examples, but the present invention is not limited by theseWorking Examples.

WORKING EXAMPLES Working Example 1 Isolation of Novel Metalloproteinase(MT-MMP) cDNA

(a) Construction of cDNA Library

Total RNA was extracted from human placenta tissue by a guanidine-cesiumchloride method (Biochemistry, 18:5294-5299, 1979) and poly(A)⁺RNA waspurified using an oligo(dT)cellulose column. Using a purifiedpoly(A)⁺RNA as a template and an oligo(dT) primer, cDNA was synthesizedaccording to the Gubler-Hoffman method (Gene, 25:263-269, 1983). Theends of the cDNA were converted to blunt end with T₄ DNA polymerase, andEcoR I sites present in the cDNA were methylated by EcoR I methylase.Using T₄ DNA ligase, an EcoR I linker [d(pG-G-A-A-T-T-C-C)] and the cDNAwere ligated, and cDNA possessing EcoR I sites at both ends wasgenerated by EcoR I digestion. Using T₄ DNA ligase, this cDNA was clonedinto EcoR I site of λgtll. In vitro packaging of this cDNA was carriedout, for example, using an in vitro packaging kit (Amersham), and a cDNAlibrary was thus constructed. A commercial cDNA library such as a humanplacenta cDNA library (Clontech) can be used as a cDNA library.

(b) Preparation of synthetic oligonucleotide primer

The sequences denoted by Sequence SEQ ID NOS:3 (P-1) and 4 (P-2) wereselected from among amino acid sequences of the known MMP family ashighly conserved amino acid sequences in the MMP family, andoligodeoxynucleotide primers corresponding respectively to oligopeptideP-1 and oligopeptide P-2 were designed. Specifically, when amino acidscoded by two or more codons were present in an oligopeptide, thesequences were designed as a mixture as shown in SEQ ID NOS:5 (primer 1)and 6 (primer 2). Primer 1 and primer 2 were synthesized by aβ-cyanoethyl phosphoamidite method using a DNA synthesizer (AppliedBiosystems Model 392). Using a NICK column (Pharmacia) equilibrated with10 mM sodium phosphate buffer, pH 6.8 the obtained primer 1 and primer 2were purified.

(c) Gene amplification by PCR

Using a human placenta-derived cDNA as a template and primers 1 and 2noted in the above section (b), a PCR (PCR Technology, Stockton Press,pp. 63-67, 1989) was run.

As a result, a 390 bp PCR product was yielded. The obtained PCR productwas cloned in an appropriate plasmid, e.g., pUC 119 or pBluescript, andthe nucleotide sequence of the PCR product was determined using afluorescence DNA sequencer (Applied Biosystems, Model 373A) and a Taqdye-primer cycle sequencing kit (Applied Biosystems). Among various PCRproducts whose nucleotide sequences were determined, PCR product Ahaving no homology to nucleotide sequences of previously reported MMPswas obtained. PCR product A was used as a probe for screening the humanplacenta cDNA library noted in the foregoing section (a). ³²P labelingof the probe was generated using a random primed DNA labeling kit(Boehringer Mannhaim).

(d) Screening of novel MMP gene from cDNA library and DNA sequencing.

Host E. coli Y1090 was transfected with the human placenta cDNA libraryconstructed in the λgtll cited in the foregoing section (a) and plaqueswere formed. Specifically, Y1090 was cultured overnight in an L brothcontaining 0.02% maltose, and bacteria were harvested and suspended in10 mM MgSO₄. This cell suspension and a phage solution were mixed,incubated at 37° C. for 15 minutes, and then the phages were adsorbedonto the host bacteria. Soft agar was added thereto, and the materialwas spread on an L plate (the above-noted operation is hereinaftertermed “plating”). The plate was incubated overnight at 42° C. and aplaque was formed, after which a nylon filter (e.g., Hibond-N, Amersham)or a nitrocellulose filter (e.g., HATF, Millipore) was placed onto theplate and left in place for approximately 30 seconds. The filter wasgently peeled and immersed in an alkaline denaturant (0.1 M NaOH, 1.5 MNaCl) for 30 seconds, then immersed in a neutralizing solution (0.5 MTris-HCl buffer, pH 8 containing 1.5 M NaCl) for 5 minutes. The filterwas then washed with 2× SSPE (0.36 M NaCl, 20 mM NaH₂PO₄, 2 mM EDTA) anddried. The foregoing plaque-to-filter transfer was repeated, and atleast two filters were prepared. However, plate contact time for thesecond and subsequent filters was extended to approximately 2 minutes.Filters were baked 2 hours at 80° C. and DNA was thus fixed. The twofilters, at a minimum, prepared from one plate were respectively washed1 hour at 42° C. in a wash solution (50 mM Tris-HCl buffer, pH 8.0containing 1 M NaCl, 1 mM EDTA and 0.1% SDS), placed in a hybridizationbag, and prehybridization was carried out by 6 to 8 hours immersion at42° C. in a prehybridization solution [50% formamide, 5×Denhardt'ssolution (0.2% bovine serum albumin, 0.2% polyvinylpyrolidone), 5× SSPE,0.1% SDS, 100 μg/ml heat-denatured salmon sperm DNA]. Next, the³²P-labeled probe noted in section (c), heat-denatured for 5 minutes at100° C., was added to the prehybridization solution, and hybridizationwas carried out overnight at 42° C. After hybridization was complete,the filters were washed at room temperature with an excess of 2× SSCsolution containing 0.1% SDS. Next, the filters were placed for 15minutes at 68° C. in 1× SSC solution containing 0.1% SDS. The filterswere then dried, layered with X-ray film (Kodak XR), and 1 weekautoradiography was then carried out at −70° C. The X-ray films weredeveloped, replica filters in duplicate produced from one plate werepiled up each other, and signals that appeared precisely same place onduplicate filters were marked. Plaques corresponding to marked signalswere suspended in SM solution (50 mM Tris-HCl buffer, pH 7.5 containing0.1 M NaCl and 10 mM MgSO₄). These phage suspensions were appropriatelydiluted and plating was performed, screening similar to that noted abovewas carried out, and recombinant phages were obtained.

(e) Preparation of recombinant λgtll DNA

Each cloned phages was plated, incubated for 3 hours at 42° C., andincubated overnight at 37° C. Several drops of chloroform was then addedto the SM solution and the material was left at room temperature for 30minutes. The SM solution together with the upper layer of soft agar wasthen scraped off, and centrifuged. Polyethylene glycol was added to a10% final concentration in the supernatant, and the material was mixedand left at 4° C. for 1 hour. The material was then centrifuged, thesupernatant was discarded, and phage particles were collected. The phageparticles were suspended in SM solution and purified by a glycerolgradient ultracentrifugation method (see “Molecular Cloning, aLaboratory Manual”, T. Maniastis et al., Cold Spring Harbor: LaboratoryPress pp. 2.78, 1989). The phages obtained were suspended in SM solutionand treated with DNase I and RNase A. A mixture of 20 mM EDTA, 50 μg/mlproteinase K, and 0.5% SDS was then added, and the material wasincubated at 65° C. for 1 hour. The material was then subjected tophenol extraction and diethylether extraction, and DNA was precipitatedby ethanol precipitation. The DNA obtained was washed with 70% ethanol,dried, and dissolved in TE solution (10 mM Tris-HCl buffer, pH 8containing lOmM EDTA).

(f) Sequencing of the insertion fragment

The μgtll DNA prepared in the above section (e) was digested with EcoRI, an insertion fragment was excised and purified, and cloned into theEcoR I site of a pBluescript (Stratagene) vector. E. coli NM522 XLI-Blue(Deposited in the National Institute of Bioscience and Human-Technologyas Deposit No. P-15032). was transformed with this recombinantpBluescript. The F′ transformed cells were selected, infected withhelper phage VCSM13 (Stratagene), and cultured overnight. The culturewas centrifuged and the bacteria were removed, and PEG/NaCl was added toprecipitate the phages. The precipitate was suspended in TE solution,and single-stranded DNA was extracted with phenol and recovered byethanol precipitation. The single-stranded DNA was sequenced using afluorescence DNA sequencer (Applied Biosystems, Model 373A) and a Taqdye-primer cycle sequencing kit (Applied Biosystems). The total lengthof the sequence determined was 3403 base pairs, and the sequence thereofis denoted by SEQ ID NO:2. The nucleotide sequence in SEQ ID NO:2 wassearched using the Genbank/EMBL DNA database, but an identical sequencedid not exist.

(g) Analysis of Gene Product

Hydrophilic and hydrophobic values of the amino acid sequence denoted bySEQ ID NO:1, as predicted from the nucleotide sequence denoted by SEQ IDNO:2, were calculated by the KyteDoolittle method (J. Mol. Biol.,157:105-132, 1982), and the hydrophilic and hydrophobic distributionplot shown in FIG. 1 was determined. A hydrophobic domain comprising asequence of 20 or more linked hydrophobic amino acids peculiar to amembrane binding protein was present from position 533 to position 562of the C-terminal region of SEQ ID NO:1, and the sequence thereof isshown in SEQ ID NO:7. Such a sequence of linked hydrophobic amino acidsdoes not exist in previously known MMPs.

When the homology of the amino acid sequence in SEQ ID NO:1 was comparedto reported MMPs amino acid sequences, the amino acid sequence in SEQ IDNO:1 showed homology with the MMP family. Specifically, processing sitebetween precursor and active enzyme and active site conserved to anextremely high degree among MMP family were each highly conserved inMT-MMP as well (SEQ ID NO:1, amino acids numbers 88-97 and 112-222).

Working Example 2 Gene Expression

(a) Expression in Tissues

Using ³²P-labeled PCR product A noted in Working Example 1, section (c)as a probe, hybridization was performed with poly(A)⁺ RNA blottedmembrane, human multiple tissue Northern Blots (Clontech), whichcontains poly(A)⁺ RNA from human heart, brain, placenta, lung, liver,skeletal muscle, kidney and pancreas. Human multiple tissue NorthernBlot filters wetted with 3× SSC (0.45 M NaCl, 0.045 M trisodiumcitrate·2H₂O, pH 7.0) were prehybridized for 2 to 3 hours in aprehybridization solution (0.75 M NaCl, 2.5 mM EDTA, 0.5× Denhardt'ssolution, 50% formamide and 20 mM Tris-HCl buffer, pH 7.5 containing 1%SDS) with gentle agitation. Next, a heat-denatured probe was added tothe hybridization solution (10% sodium dextran and 50 μg/ml denaturedsalmon sperm DNA-containing prehybridization solution), theprehybridization solution was replaced, and hybridization was performedovernight at 43° C. After hybridization was complete, the filters werewashed with 2× SSC containing 0.1% SDS. Next, the filters were placedfor 15 minutes at 68° C. in 1× SSC containing 0.1% SDS. The filters werethen dried, layered with X-ray film (Kodak XR), and 1 weekautoradiography was then carried out at −70° C. The size of the MT-MMPgene transcripts was 4.8 kb in each tissue. When the developed X-rayfilms were traced by a densitometer and signal intensity was measured,among the investigated tissues, MT-MMP genes were found to be highlyexpressed in the lung, placenta and kidney.

(b) Expression in Tumour Tissues

Normal and tumour tissues were taken from samples of two squamous cellcarcinomas human lung, respectively, and total RNA was extracted by aguanidine-cesium chloride method. 10 μg of each said RNA was applied to1% agarose electrophoresis and then transferred onto a nylon membrane.Hybridization was then carried out with the ³²P-labeled probe noted inWorking Example 1, section (c). Hybridization and autoradiographytracing were performed as described in the foregoing section (a). Ineach human lung squamous cell carcinoma, significantly higher expressionwere seen in tumour tissue (see FIG. 4 T) than in normal tissue (seeFIG. 4 N).

Working Example 3 Preparation of Monoclonal Antibodies

(a) Preparation of Polypeptides as Antigen

From the MT-MMP amino acid sequence denoted by SEQ ID NO:1, sequencesdenoted by SEQ ID NOS:8, 9 and 10 (sequence of SEQ ID NO:1 amino acidnumbers 160-173, 320-333, and 498-512, respectively; hereinafter termedpolypeptide A, polypeptide B and polypeptide C, respectively) wereselected as specific sequences having low homology to other members ofMMP family. These polypeptides were synthesized by Fmoc-BOP method usinga peptide synthesizer (MilliGen/Biosearch, Peptide Synthesizer 9600),and cysteine was introduced at the N-terminus. Each synthesized peptidewas purified by high speed liquid chromatography.

(b) Preparation of Each Polypeptides and Keyhole Limpet HemocyaninComplexes

2 mg of keyhole limpet hemocyanin (KLH) dissolved in 1 ml of 0.1 Mphosphate buffer, pH 7.5 and 1.85 mgN-(ε-maleimidocaproyloxy)succinimide dissolved in 200 μldimethylformamide were mixed and incubated at 30° C. for 30 minutes.Next, the above-noted mixture was applied to gel filtration by PD-10(Pharmacia) equilibrated with 0.1 M phosphate buffer, pH 7.0. KLH-boundmaleimide was collected and concentrated to less than 1.5 ml. Eachpolypeptide synthesized in the foregoing section (a) was respectivelydissolved in 1 ml of 0.1 M phosphate buffer, pH 7.0 and mixed withKLH-bound maleimide at a molar ratio representing a factor of 50. Thismaterial was then incubated 20 hours at 4° C., and KLH-polypeptidecomplexes were thus prepared.

(c) Preparation of Antibody-producing Cells

As an initial immunization, eight-week-old Balb/c female mice were givenan intraperitoneal administration of 250 μg of a complex of KLH and,respectively, polypeptide A, polypeptide B or polypeptide C prepared inthe above section (b), in Freund's complete adjuvant. After 18 days, therespectively immunized mice were boosted intraperitoneally with 200 μgof the respective complexes dissolved in 0.1 M phosphate buffer, pH 7.5.After 32 days, a final immunization of 100 μg of each complex wasadministered intravenously as the booster immunization. Three daysthereafter, splencytes were extirpated and splencyte suspensions wereprepared.

(d) Cell Fusion

Fusion with 8-azaguanine-resistant myeloma cell SP2 (SP2/O-Ag14) wasperformed according to a modifying method of Oi et al (see SelectedMethods in Cellular Immunology, Mishell, B. B. and Shiigi, S. M., ed.,W. H. Freeman and Company pp. 351-372, 1980). Fusion of myeloma cell SP2with karyo-splencytes from mice immunized with the polypeptide AKLHcomplex is discussed in details, hereafter.

Through the following procedures, karyo-splencytes prepared in theforegoing section (c) (cell viability 100%) were fused in a 5:1 ratiowith myeloma cells (cell viability 100%). A polypeptide A-immunizedsplencyte suspension and myeloma cells were separately washed in RPMI1640 medium. The material was then suspended in the same medium, and3×10⁸ cells of karyo-splencytes and 6×10⁷ cells of myeloma cells weremixed for fusion. The cells were then precipitated by centrifugation,and all the supernatant was completely discarded by suction. 2.0 ml ofPEG 4000 solution (RPMI 1640 medium containing 50% [w/v] polyethyleneglycol 4000) prewarmed at 37° C. was added dropwise to the precipitatedcells over 1 minute, 1 minute stirring was performed, and the cells wereresuspended and dispersed. Next, 2.0 ml of RPMI 1640 medium prewarmed at37° C. was added in a dropwise fashion over 1 minute. After repeatingthe same operation once more, 14 ml of RPMI 1640 medium was addeddropwise over 2 to 3 minutes under constant stirring, and the cells weredispersed. The dispersion was centrifuged and the supernatant wascompletely discarded by suction. Next, 30 ml of NS-1 medium (RPMI 1640medium containing filter-sterilized 15% [w/v] fetal calf serum [JRHBiosciences]) prewarmed at 37° C. was rapidly added to the precipitatedcells, and the large cell clumps were carefully dispersed by pipetting.The dispersion was then diluted by adding 30 ml of NS-1 medium, and6.0×10⁵ cells/0.1 ml/well was added to a polystyrene 96-microwell plate.The above-noted cell-filled microwells were cultured in 7% carbonic acidgas/93% atmospheric air at 37° C. and 100% humidity.

In the case of splencytes derived from mice immunized with thepolypeptide B-KLH complex, 6.4×10⁸ cells of splencytes and 1.28×10⁸cells of myeloma cells were mixed, and respectively, 4.3 ml, 38.7 ml and129 ml of the above-used PEG 4000 solution, RPMI 1640 medium and NS-1medium were used. In the case of splencytes derived from mice immunizedwith the polypeptide C-KLH complex, 6.8×10⁸ cells of splencytes and1.36×10⁸ cells of myeloma cells were mixed, and 4.5 ml, 40.5 ml and 135ml of respectively PEG 4000 solution, RPMI 1640 medium and NS-1 mediumwere used.

(e) Selective Amplification of Hybridomas by Selective Culture Medium

On the day following the start of culturing mentioned in the abovesection (d) (Day 1), 2 drops (approx. 0.1 ml) HAT culture medium (100 μMhypoxanthine, 0.4 μM aminopterin and 16 μM thymidine added to NS-1culture medium) were added to the cells with a Pasteur pipette. On Days2, 3, 5 and 8, half of each culture medium (approx. 0.1 ml) was replacedwith fresh HAT medium, and on Day 11, half of each culture medium wasreplaced with fresh HT culture medium (HAT culture medium not containingaminopterin). On Day 14, for all the wells in which hybridoma growth wasobserved to the naked eye, positive wells were investigated byenzyme-linked immunoadsorbent assay (ELISA). Specifically, thepolystyrene 96-well plate was respectively coated with polypeptides A, Band C serving as antigens, washed using PBS for washing (containing0.05% Tween 20), and unadsorbed peptides were thus removed. In addition,the uncoated portion of each well was blocked with 1% BSA. 0.1 ml ofsupernatant from wells in which hybridoma growth was confirmed was addedto each polypeptide-coated well, and the plate was stood at roomtemperature for approximately 1 hour.

Horseradish peroxidase-labeled goat anti-mouse immunoglobulin was addedas a secondary antibody, and the plate was again stood at roomtemperature for approximately another 1 hour. A substrate of hydrogenperoxide and o-phenylenediamine was added, and the degree of colordevelopment was measured as absorbance at 492 nm using a microplatelight absorbency measuring device (MRP-A4, Tosoh).

(f) Hybridoma Cloning

Hybridomas in wells positive with respect to individual antigenpeptides, as obtained in the foregoing section (e), were monoclonedaccording to the limiting dilution method. Specifically, hybridomas werediluted to 5, 1 and 0.5 per well and were respectively added to 36, 36and 24 wells of a 96 microwells. On Day 5 and Day 12, approximately 0.1ml NS-1 medium was added to each well. Approximately 2 weeks aftercloning began, the ELISA noted in section (e) was performed for groupsin which sufficient hybridoma growth was visually confirmed and 50% ormore wells were negative for colony formation. If all tested wells werenot positive, 4 to 6 antibody-positive wells in which the number ofcolonies was 1 were selected, and recloning was performed. Finally, asshown in Table 1 and Table 2, 12, 20 and 9 hybridomas were obtainedwhich produced monoclonal antibodies against polypeptide A, polypeptideB or polypeptide C, respectively.

(g) Hybridoma Culturing and Monoclonal Antibody Purification

Each obtained hybridoma was cultured in NS-1 medium and a 10 to 100μg/ml concentration of monoclonal antibody was successfully obtainedfrom the supernatant thereof. In addition, BALB/c mice given an one weekprior intraperitoneal administration of pristane were given a similarintraperitoneal administration of 1×10⁷ cells of obtained hybridomas,and after 1 to 2 weeks, abdominal fluid containing 4 to 7 mg/ml ofmonoclonal antibody was successfully obtained. The abdominal fluidobtained was salted out by 40% saturated ammonium sulfate, and IgG classantibodies were adsorbed to Protein A Affigel (Bio-Rad) and purified byelution with a 0.1M citric acid buffer, pH 5.

(h) Determination of Monoclonal Antibody Class and Subclass

In accordance with the above-discussed ELISA, the supernatant ofmonoclones obtained in section (f) were added to microtitration platesrespectively coated with polypeptide A, polypeptide B or polypeptide C.After washing with PBS, isotype-specific rabbit anti-mouse IgGantibodies (Zymed Lab.) were added. After washing with PBS, horseradishperoxidase-labeled goat anti-rabbit IgG (H+L) was added, and class andsubclass were determined using hydrogen peroxide and2.2′-azino-di(3-ethylbenzthiazolinic acid) as a substrate.

(i) Specificity of Anti-MT-MMP Monoclonal Antibodies

The cross-reactivity of five varieties of anti-MT-MMP monoclonalantibodies (monoclone numbers 113-5B7, 113-15E7, 114-lF1, 114-2F2 and118-3B1) exhibiting a positive reaction against a human MT-MMP peptidewas determined by the ELISA noted in the foregoing section (e), using asrespective antigens: proMMP-1 (Clin. Chim. Acta, 219:1-14, 1993),proMMP-2 (Clin. Chim. Acta, 221:91-103, 1993) and proMMP-3 (Clin. Chim.Acta, 211:59-72, 1992) respectively purified from the supernatant ofnomal human skin fibroblast (NB1RGB) culture; proMMP-7 purified from thesupernatant of human rectal carcinoma cell (CaR-1) culture (Cancer Res.,50:7758-7764, 1990), proMMP-8 purified from human neutrophils (Biol.Chem. Hoppe-Seyler, 371 supp:295-304, 1990) and proMMP-9 purified fromthe supernatant of human fibrosarcoma cells (HT1080) culture (J. Biol.Chem., 267: 21712-21719, 1992).

Specifically, using a polystyrene 96-well plate, each well was coated byadding 50 ng/well of purified MMP-1, MMP2, MMP-3, MMP-7, MMP-8 andMMP-9, respectively. Washing was performed with PBS for washing andnon-adsorbed antigen was removed, and the uncoated portion of each wellwas blocked with PBS containing 3% skim milk. 1 μg/well of each MT-MMPmonoclonal antibody was respectively added to each well and stood atroom temperature for approximately 1 hour. After washing plate,peroxidase-labeled goat anti-mouse immunoglobulin was added as asecondary antibody, and the plate was again stood at room temperaturefor approximately 1 hour. A substrate of hydrogen peroxide ando-phenylene diamine was added, and the degree of color development wasmeasured absorbance at 492 nm using a microplate light absorbencymeasuring device (MRP-A4, Tosoh).

In results, as shown in Table 3, each anti-MT-MMP monoclonal antibodyshowed no reactivity against purified MMPs other than the MT-MMPsupplied for testing.

TABLE 1 Polypeptide Monoclone No. Subclass/Chain A 114-1F2 γ1/κ 114-2F2γ1/κ 114-3H7 γ1/κ 114-5E4 γ1/κ 114-6G6 γ1/κ 114-8D10 γ1/κ 114-9H3 μ/κ114-15E8 γ1/κ 114-16C11 γ1/κ 114-18E4 γ1/κ 114-19F11 γ1/κ 114-20H5 μ/κ B113-1E3 y3/κ 113-2E9 y3/κ 113-3F6 y2b/κ 113-4H7 y3/κ 113-5B7 y3/κ113-7C6 y2b/κ 113-9G9 y3/κ 113-10F2 y3/κ 113-13G11 y3/κ 113-15E7 y3/κ113-16H8 y3/κ 113-17G12 μ/κ 113-19A10 μ/κ 113-20G11 y3/κ 113-21H3 y1/κ113-26D3 μ/κ 113-44C1 y1/κ 113-46B7 y1/κ 113-53G5 μ/κ 113-63E8 γ1/κ

TABLE 2 Polypeptide Monoclone No. Subclass/Chain C 118-3B1 γ2b/κ 118-6F3γ2b/κ 118-8D11 γ1/κ 118-9B11 γ1/κ 118-13D11 α/κ 118-18C12 γ1/κ 118-20A3y2b/κ 118-25C3 γ1/κ 118-26F5 γ3/κ

TABLE 3 Monoclone Cross reactivity No. MMP-1 MMP-2 MMP-3 MMP-7 MMP-8MMP-9 113-5B7 — — — — — — 113-15E7 — — — — — — 114-1F2 — — — — — —114-2F2 — — — — — — 118-3B1 — — — — — — —: No reaction

Working Example 4 Expression and Identification of Gene Product

By means of EcoR I cleavage, an insertion fragment was excised from therecombinant pBluescript containing a cloned MT-MMP gene, constructed insection (f) of Working Example 1. Cloning was then carried out at anEcoR I site of the eukaryotic expression vector pSG5 (Stratagene) Then,human fibrosarcoma cells (HT1080) were transfected with said recombinantpSG5 by a calcium phosphate method. Specifically, 20 μg of recombinantpSG5 and 62 μl of 2M CaCl₂ was added to distilled water, and 2× HBSPsolution (50 mM HEPES buffer, pH 7.1 containing 1.5 mM Na₂HPO₄, 10 mMKCl, 280 mM NaCl and 12 mM glucose) was added to the bottom of the tubeto form a total volume of 1 ml. This material was mixed, stood at roomtemperature for approximately 30 minutes, and thorough precipitateformation was carried out. The precipitate was dispersed by pipetting,added dropwise to HT1080 cells and incubated for approximately 4 hoursin a CO₂ incubator. Next, the culture medium was removed, a 15% glycerolsolution was added and treated for 1 to 3 hours, the glycerol wasdiscarded by suction, washed with PBS and fresh culture mediumcontaining ³⁵S-methionine was added. Culturing was continued, andcellular proteins were labeled by ³⁵S. Incidentally, expression ofMT-MMP genes in HT1080 cells cannot be detected by Northern Blotanalysis.

The cells were incubated for 1 hour at 4° C. in a lysing buffer solution(0.01 M Tris-HCl buffer, pH 8 containing 1% Triton X-100, 1% bovinehemoglobin, 1 mM iodoacetamide, 0.2U trypsin inhibitor, 1 mM PMSF and0.14 M NaCl). The cell lysate was centrifuged and the supernatant wasrecovered. Sepharose-4B (Pharmacia) coupled with a monoclonal antibodyobtained in Working Example 3 was added to the supernatant, the materialwas incubated at 4° C. for 2 hours with agitation, andimmunoprecipitation was carried out. Monoclonal antibodies againstpolypeptide A used in immunoprecipitation were two of the 12 obtained inWorking Example 3 which had low non-specific reactivity (monoclonenumbers 114-1F2 and 114-2F2 [Assignment No. FERM BP-4743]). Next,Sepharose 4B coupled with monoclonal antibodies subjected toimmunoprecipitation were precipitated by centrifugation, washed threetimes with a washing solution (0.01 M Tris-HCl buffer, pH 8 containing1% Triton X-100, 1% bovine hemoglobin and 0.14 M NaCl), and lastly,washed with 0.05 M Tris-HCl buffer, pH 6.8. A sample buffer for SDSpolyacrylamide electrophoresis was added to washed Sepharose-4B coupledwith a monoclonal antibody, boiled 5 minutes at 100° C., and SDSpolyacrylamide electrophoresis was carried out. The electrophoresed gelwas layered with X-ray film (Kodak XR), 1 week autoradiography was thencarried out at −70° C., and the developed X-ray films were traced by adensitometer to measure signal intensity. With each of the anti-MT-MMPmonoclonal antibodies used (monoclone numbers 114-1F2 and 114-2F2), theimmunoprecipitate contained a 63 kDa protein. In cells transfected witha pSG5 vector alone not containing an MT-MMP gene as a control,anti-MTMMP monoclonal antibodies (monoclone numbers 114-1F2 and 114-2F2)did not precipitate a 63 kDa protein. The 63 kDa molecular weight of theprotein detected by immunoprecipitation nearly matched a molecularweight of 65.78 kDa calculated from the amino acid sequence denoted bySEQ ID NO:1. In addition, a variant MT-MMP expression plasmid wasconstructed in which amino acids from position 13 to position 101 weredeleted from the amino acid sequence denoted by SEQ ID NO:1, HT1080cells was transfected with said variant as stated above, andimmunoprecipitation was carried out. With HT1080 cells to which thevariant MT-MMP gene was introduced, a 63 kDa protein was not detected,and a 55 kDa protein was detected. This molecular weight matched amolecular weight predicted from the introduced deletion.

EXPERIMENTAL EXAMPLE

(a) Activation of proMMP-2 by MT-MMP Expression

Recombinant pSG5 carrying a cloned MT-MMP gene, constructed in WorkingExample 4, and a pSG5 vector alone, serving as a control, similarlytransfected into HT1080 cells by the calcium phosphate method mentionedin Working Example 4, or into mouse embryonic fibroblasts NIH3T3.However, a regular fresh culture medium was used in lieu of the freshculture medium containing ³⁵S-methionine. Both the HT1080 cells and theNIH3T3 cells secreted proMMP-2 and proMMP-9 (corresponding respectivelyto the 66 kDa and 97.4 kDa bands in FIG. 6), and in cells transfectedwith an MT-MMP gene, MT-MMP expression was confirmed byimmunoprecipitation experiments (See Working Example 4).

The transfectants obtained were cultured for 24 hours in a serum freemedium and the recovered culture supernatant was supplied forzymography. The culture supernatant was mixed with an SDS polyacrylamideelectrophoresis buffer (non-reducing condition) and left at 4° C.overnight. Electrophoresis was then performed at 4° C., with a 20 mAcurrent, using a 10% polyacrylamide gel containing 1 mg/ml casein. Afterelectrophoresis, the gel was washed with a gelatinase-buffer (Tris-HClbuffer, pH 7.6 containing 5 mM CaCl₂ and 1 μM ZnSO₄) containing 2.5%Triton X-100 with gentle agitation for 15 minutes, and this operationwas repeated twice. Next, the gel was immersed in a gelatinase-buffercontaining 1% Triton X-100 and stood at 37° C. overnight. The buffer wasdiscarded and the gel was stained for 1 hour with 0.02% CoomassieBrilliant Blue-R (dissolved in 50% methanol/10% acetic acid) anddestained by immersion in a destaining solution (5% methanol, 7.5%acetic acid).

As shown in FIG. 6, MT-MMP gene-transfected HT1080 cells produced new 64kDa and 62 kDa bands, confirming proMMP-2 activation. This active-formMMP-2 exhibited the same molecular weight as an active-form MMP-2molecule induced by treatment of cells with 100 μg/ml of concanavalin Aand reacted specifically against anti-MMP-2 monoclonal antibodies. Thisactivation was not observed in a control transfected with a vectoralone. Likewise, proMMP-9 showed no change in molecular weight and noactivation similar to that observed in control cells. Such activation ofproMMP-2 depending on MT-MMP expression was also observed in MT-MMPgene-transfected NIH3T3 cells.

(b) Activation of ProMMP-2 by MT-MMP Expression Cell Membrane Fraction

In a manner similar to that noted in the above section (a), Africangreen monkey kidney-derived COS-1 cells were transfected withrecombinant pSG5 containing cloned MT-MMP gene, or with control pSG5vector alone by a calcium phosphate method. A cell membrane fraction wasthen prepared from the obtained transfectant according to the method ofStrongin et al. (J. Biol. Chem., 268:14033-14039, 1993).

The transfectant was washed with PBS, and cells were harvested bycentrifugation and suspended in a 25 mM Tris-HCl buffer, pH 7.4containing 8.5% sucrose, 50 mM NaCl, 10 mM Nethylmaleimide, 10 μg/mlaprotinin, 1 μg/ml pepstatin A, 1 μg/ml leupeptin and 1 mMphenylmethylsulfonyl fluoride. The cell suspension was homogenized in aDounce homogenizer, and the homogenate was centrifuged (3000× g, 10min., 4° C.). The resulting supernatant was ultracentrifuged (100,000×g, 2 hours) and the precipitate was suspended in a 25mM TrisHCl buffer,pH 7.4 containing 50 mM NaCl, 10 mM N-ethylmaleimide, 10 μg/mlaprotinin, 1 μg/ml pepstatin A, 1 μg/ml leupeptin and 1 mMphenylmethylsulfonyl fluoride. This suspension was fractionated bydiscontinuous sucrose density gradient centrifugation (20, 30, 50, 60%sucrose solutions; 100,000× g; 2 hours; 4° C.), and bands of cellmembrane fractions appeared were recovered. These fractions wereprecipitated again by ultracentrifugation (100,000x g, 2 hours),suspended in 25mM HEPES/KOH buffer, pH 7.5 containing 0.lmM CaCl₂ and0.25% Triton X-100, and adjusted to a final protein concentration of 1-2mg/ml. This suspension was ultracentrifuged (100,000x g, 1.5 hours, 4°C.) to remove insoluble residue, and the supernatant obtained was takenas a cell membrane fraction.

Cell membrane fractions (protein content 20 μg) respectively preparedfrom untreated COS-1 cells or from COS-1 cells transfected with pSG5vector alone or pSG5 vector with an MT-MMP gene were incubated withHT1080 cell culture supernatant at 37° C. for 2 hours. Using thesesamples, the zymography noted in the above section (a) was performed.

In the results, new 64 kDa and 62 kDa bands appeared and the activationof proMMP-2 present in HT1080 cell culture supernatant was observed onlywhen cell membrane fractions derived from MT-MMP gene-transfected COS-1cells were used (see FIG. 7), and the activation of proMMP-2 wasinhibited by the addition of recombinant (r) human TIMP-2. These resultsexhibited the activation of proMMP-2 by MT-MMP expressed on a cellmembrane.

(c) Stimulation of cellular invasion in vitro due to MT-MMP expression

Invasion of cells was assayed by modified Boyden Chamber method (CancerRes., 47:3239-3245, 1987), and operations were carried out in accordancewith the manufucture's instructions for a Biocoat Matrigel InvasionChamber (Becton Dickinson).

In a manner similar to that noted in the foregoing section (a), HT1080cells or NIH3T3 cells were transfected with recombinant pSG5 carrying acloned MT-MMP gene, or a control pSG5 vector alone, by a calciumphosphate method, and each of these host cells secreted proMMP-2. Theresulting transfectants were then suspended in DMEM medium containing0.1% BSA, and 2×10⁵ cells were seeded onto an uncoated filter (pore size8 μm) or a preswelled Matrigel Coat filter in a Biocoat MatrigelInvasion Chambers. After 24 hours incubation in a CO₂ incubator at 37°C., the filters were fixed by 10 seconds immersion in methanol. Thefilters were then stained by hematoxylin for 3 minutes, washed, andstained by eosin for 10 seconds, and the number of cells invaded thebottom surface of the filters were counted under a light microscope (ata magnification of ×400).

In the MT-MMP gene-transfected HT1080 cells and NIH3T3 cells, more thantwice as many invading cells were seen compared to cells transfectedwith the control vector alone (See FIG. 8 Matrigel). Specifically,MT-MMP expression was seen to stimulate cellular invasion. Furthermore,the addition of 10 μg/ml of r-human TIMP-2 to this assay system clearlysuppressed cellular invasion (see FIG. 8 Matrigel+r-human TIMP-2).

BRIEF DESCRIPTION OF THE FIGURES

FIG. 1 shows hydrophilic and hydrophobic distribution diagrams for theamino acid sequence of MT-MMP, according to the Kyte-Doolittle method.

FIGS. 2A, 2B, 2C, 2D, 2E, 2F, 2G and 2H are figures comparing sequentialhomology between the amino acid sequences of MT-MMPn SEQ ID NO:1 andthose of the known MMP family (MMP1 (SEQ ID NO:12), MMP-2 (SEQ IDNO:17), MMP-3 (SEQ ID NO:15), MMP-7 (SEQ ID NO:18), MMP-8 (SEQ IDNO:13), MMP-9 (SEQ ID NO:16), MMP-10 and MMP-11) Letters in each figureindicate respective amino acids; A corresponding to Ala, C to Cys, D toAsp, E to Glu, F to Phe, G to Gly, H to His, I to Ile, K to Lys, L toLeu, M to Met, N to Asn, P to Pro, Q to Gln, R to Arg, S to Ser, T toThr, V to Val, W to Trp and Y to Tyr. FIGS. 2A through 2H are anintegral unit and comprise a single figure.

FIG. 3 shows a relative expression of MT-MMP mRNA in various humantissues, according to Northern blot analysis.

FIG. 4 shows a relative expression of MT-MMP mRNA in a normal tissue anda tumour tissue of two samples of human lung squamous cell carcinoma,according to Northern blot analysis.

FIG. 5 shows results for detection, by immunoprecipitation, of MT-MMPproteins expressed in HT1080 cells transfected with MT-MMP cDNA. Thefigure shows a scan by a densitometer, and the darkened areas indicatethe location of MT-MMP immunoprecipitated by anti-MT-MMP monocionalantibody.

FIG. 6 shows an activation of proMMP-2 by expression of MT-MMP,according to zymography of culture supernatant from HT1080 and NIH3T3cells transfected with MT-MMP cDNA.

FIG. 7 shows an activation of proMMP-2 by a cell membrane fraction ofCOS-1 cells transfected with MT-MMP cDNA, according to zymography.

FIG. 8 shows a stimulation of the cellular invasion by expression ofMT-MMP, according to a partially modified Boyden chamber method.

19 1 582 PRT Homo sapiens 1 Met Ser Pro Ala Pro Arg Pro Ser Arg Cys LeuLeu Leu Pro Leu Leu 1 5 10 15 Thr Leu Gly Thr Ala Leu Ala Ser Leu GlySer Ala Gln Ser Ser Ser 20 25 30 Phe Ser Pro Glu Ala Trp Leu Gln Gln TyrGly Tyr Leu Pro Pro Gly 35 40 45 Asp Leu Arg Thr His Thr Gln Arg Ser ProGln Ser Leu Ser Ala Ala 50 55 60 Ile Ala Ala Met Gln Lys Phe Tyr Gly LeuGln Val Thr Gly Lys Ala 65 70 75 80 Asp Ala Asp Thr Met Lys Ala Met ArgArg Pro Arg Cys Gly Val Pro 85 90 95 Asp Lys Phe Gly Ala Glu Ile Lys AlaAsn Val Arg Arg Lys Arg Tyr 100 105 110 Ala Ile Gln Gly Leu Lys Trp GlnHis Asn Glu Ile Thr Phe Cys Ile 115 120 125 Gln Asn Tyr Thr Pro Lys ValGly Glu Tyr Ala Thr Tyr Glu Ala Ile 130 135 140 Arg Lys Ala Phe Arg ValTrp Glu Ser Ala Thr Pro Leu Arg Phe Arg 145 150 155 160 Glu Val Pro TyrAla Tyr Ile Arg Glu Gly His Glu Lys Gln Ala Asp 165 170 175 Ile Met IlePhe Phe Ala Glu Gly Phe His Gly Asp Ser Thr Pro Phe 180 185 190 Asp GlyGlu Gly Gly Phe Leu Ala His Ala Tyr Phe Pro Gly Pro Asn 195 200 205 IleGly Gly Asp Thr His Phe Asp Ser Ala Glu Pro Trp Thr Val Arg 210 215 220Asn Glu Asp Leu Asn Gly Asn Asp Ile Phe Leu Val Ala Val His Glu 225 230235 240 Leu Gly His Ala Leu Gly Leu Glu His Ser Ser Asp Pro Ser Ala Ile245 250 255 Met Ala Pro Phe Tyr Gln Trp Met Asp Thr Glu Asn Phe Val LeuPro 260 265 270 Asp Asp Asp Arg Arg Gly Ile Gln Gln Leu Tyr Gly Gly GluSer Gly 275 280 285 Phe Pro Thr Lys Met Pro Pro Gln Pro Arg Thr Thr SerArg Pro Ser 290 295 300 Val Pro Asp Lys Pro Lys Asn Pro Thr Tyr Gly ProAsn Ile Cys Asp 305 310 315 320 Gly Asn Phe Asp Thr Val Ala Met Leu ArgGly Glu Met Phe Val Phe 325 330 335 Lys Lys Arg Trp Phe Trp Arg Val ArgAsn Asn Gln Val Met Asp Gly 340 345 350 Tyr Pro Met Pro Ile Gly Gln PheTrp Arg Gly Leu Pro Ala Ser Ile 355 360 365 Asn Thr Ala Tyr Glu Arg LysAsp Gly Lys Phe Val Phe Phe Lys Gly 370 375 380 Asp Lys His Trp Val PheAsp Glu Ala Ser Leu Glu Pro Gly Tyr Pro 385 390 395 400 Lys His Ile LysGlu Leu Gly Arg Gly Leu Pro Thr Asp Lys Ile Asp 405 410 415 Ala Ala LeuPhe Trp Met Pro Asn Gly Lys Thr Tyr Phe Phe Arg Gly 420 425 430 Asn LysTyr Tyr Arg Phe Asn Glu Glu Leu Arg Ala Val Asp Ser Glu 435 440 445 TyrPro Lys Asn Ile Lys Val Trp Glu Gly Ile Pro Glu Ser Pro Arg 450 455 460Gly Ser Phe Met Gly Ser Asp Glu Val Phe Thr Tyr Phe Tyr Lys Gly 465 470475 480 Asn Lys Tyr Trp Lys Phe Asn Asn Gln Lys Leu Lys Val Glu Pro Gly485 490 495 Tyr Pro Lys Ser Ala Leu Arg Asp Trp Met Gly Cys Pro Ser GlyGly 500 505 510 Arg Pro Asp Glu Gly Thr Glu Glu Glu Thr Glu Val Ile IleIle Glu 515 520 525 Val Asp Glu Glu Gly Gly Gly Ala Val Ser Ala Ala AlaVal Val Leu 530 535 540 Pro Val Leu Leu Leu Leu Leu Val Leu Ala Val GlyLeu Ala Val Phe 545 550 555 560 Phe Phe Arg Arg His Gly Thr Pro Arg ArgLeu Leu Tyr Cys Gln Arg 565 570 575 Ser Leu Leu Asp Lys Val 580 2 3403DNA Homo sapiens 2 agttcagtgc ctaccgaaga caaaggcgcc ccgagggagtggcggtgcga ccccagggcg 60 tgggcccggc cgcggagcca cactgcccgg ctgacccggtggtctcggac catgtctccc 120 gccccaagac cctcccgttg tctcctgctc cccctgctcacgctcggcac cgcgctcgcc 180 tccctcggct cggcccaaag cagcagcttc agccccgaagcctggctaca gcaatatggc 240 tacctgcctc ccggggacct acgtacccac acacagcgctcaccccagtc actctcagcg 300 gccatcgctg ccatgcagaa gttttacggc ttgcaagtaacaggcaaagc tgatgcagac 360 accatgaagg ccatgaggcg cccccgatgt ggtgttccagacaagtttgg ggctgagatc 420 aaggccaatg ttcgaaggaa gcgctacgcc atccagggtctcaaatggca acataatgaa 480 attactttct gcatccagaa ttacaccccc aaggtgggcgagtatgccac atacgaggcc 540 attcgcaagg cgttccgcgt gtgggagagt gccacaccactgcgcttccg cgaggtgccc 600 tatgcctaca tccgtgaggg ccatgagaag caggccgacatcatgatctt ctttgccgag 660 ggcttccatg gcgacagcac gcccttcgat ggtgagggcggcttcctggc ccatgcctac 720 ttcccagggc ccaacattgg aggagacacc cactttgactctgccgagcc ttggactgtc 780 aggaatgagg atctgaatgg aaatgacatc ttcctggtggctgtgcacga gctgggccat 840 gccctggggc tcgagcattc cagtgacccc tcggccatcatggcaccctt ttaccagtgg 900 atggacacgg agaattttgt gcttcccgat gatgaccgccggggcatcca gcaactttat 960 gggggtgagt cagggttccc caccaagatg ccccctcaacccaggactac ctcccggcct 1020 tctgttcctg ataaacccaa aaaccccacc tatgggcccaacatctgtga cgggaacttt 1080 gacaccgtgg ccatgctccg aggggagatg tttgtcttcaagaagcgctg gttctggcgg 1140 gtgaggaata accaagtgat ggatggatac ccaatgcccattggccagtt ctggcggggc 1200 ctgcctgcgt ccatcaacac tgcctacgag aggaaggatggcaaattcgt cttcttcaaa 1260 ggagacaagc attgggtgtt tgatgaggcg tccctggaacctggctaccc caagcacatt 1320 aaggagctgg gccgagggct gcctaccgac aagattgatgctgctctctt ctggatgccc 1380 aatggaaaga cctacttctt ccgtggaaac aagtactaccgtttcaacga agagctcagg 1440 gcagtggata gcgagtaccc caagaacatc aaagtctgggaagggatccc tgagtctccc 1500 agagggtcat tcatgggcag cgatgaagtc ttcacttacttctacaaggg gaacaaatac 1560 tggaaattca acaaccagaa gctgaaggta gaaccgggctaccccaagtc agccctgagg 1620 gactggatgg gctgcccatc gggaggccgg ccggatgaggggactgagga ggagacggag 1680 gtgatcatca ttgaggtgga cgaggagggc ggcggggcggtgagcgcggc tgccgtggtg 1740 ctgcccgtgc tgctgctgct cctggtgctg gcggtgggccttgcagtctt cttcttcaga 1800 cgccatggga cccccaggcg actgctctac tgccagcgttccctgctgga caaggtctga 1860 cgcccatccg ccggcccgcc cactcctacc acaaggactttgcctctgaa ggccagtggc 1920 agcaggtggt ggtgggtggg ctgctcccat cgtcccgagccccctccccg cagcctcctt 1980 gcttctctct gtcccctggc tggcctcctt caccctgaccgcctccctcc ctcctgcccc 2040 ggcattgcat cttccctaga taggtcccct gagggctgagtgggagggcg gccctttcca 2100 gcctctgccc ctcaggggaa ccctgtagct ttgtgtctgtccagccccat ctgaatgtgt 2160 tgggggctct gcacttgaag gcaggaccct cagacctcgctggtaaaggt caaatggggt 2220 catctgctcc ttttccatcc cctgacatac cttaacctctgaactctgac ctcaggaggc 2280 tctggggaac tccagccctg aaagccccag gtgtacccaattggcagcct ctcactactc 2340 tttctggcta aaaggaatct aatcttgttg agggtagagaccctgagaca gtgtgagggg 2400 gtggggactg ccaagccacc ctaagacctt gggaggaaaactcagagagg gtcttcgttg 2460 ctcagtcagt caagttcctc ggagatcttc ctctgcctcacctaccccag ggaacttcca 2520 aggaaggagc ctgagccact ggggactaag tgggcagaagaaacccttgg cagccctgtg 2580 cctctcgaat gttagccttg gatggggctt tcacagttagaagagctgaa accaggggtg 2640 cagctgtcag gtagggtggg gccggtggga gaggcccgggtcagagccct gggggtgagc 2700 cttaaggcca cagagaaaga accttgccca aactcaggcagctggggctg aggcccaaag 2760 gcagaacagc cagagggggc aggaggggac caaaaaggaaaatgaggacg tgcagcagca 2820 ttggaaggct ggggcccggc agccaggtta aagctaacagggggccatca gggtgggctt 2880 gtggagctct caggaagggc cctgaggaag gcacacttgctcctgttggt ccctgtcctt 2940 gctgcccagg cagggtggag gggaagggta gggcagccagagaaaggagc agagaaggca 3000 cacaaacgag gaatgagggg cttcacgaga ggccacagggcctggctggc cacgctgtcc 3060 cggcctgctc accatctcag tgagggacag gagctggggctgcttaggct gggtccacgc 3120 ttccctggtg ccagcacccc tcaagcctgt ctcaccagtggcctgccctc tcgctccccc 3180 acccagccca cccattgaag tctccttggg tcccaaaggtgggcatggta ccggggactt 3240 gggagagtga gacccagtgg agggagcaag aggagagggatgtggggggg tggggcacgg 3300 gtaggggaaa tggggtgaac ggtgctggca gttcggctagatttctgtct tgtttgtttt 3360 tttgttttgt ttaatgtata tttttattat aattattatatat 3403 3 7 PRT Unknown Description of Unknown Organism Highlyconserved sequence fragments from MMP family 3 Pro Arg Cys Gly Val ProAsp 1 5 4 9 PRT Unknown Description of Unknown Organism Highly conservedsequence fragments from MMP family 4 Gly Asp Ala His Phe Asp Asp Asp Glu1 5 5 20 DNA Artificial Sequence Description of Artificial SequenceSynthetic DNA 5 ccmmgvtgys gvrwbccwga 20 6 25 DNA Artificial SequenceDescription of Artificial Sequence Synthetic DNA 6 ytcrtsvtcr tcraartgrrhrtcy 25 7 30 PRT Homo sapiens 7 Gly Gly Gly Ala Val Ser Ala Ala Ala ValVal Leu Pro Val Leu Leu 1 5 10 15 Leu Leu Leu Val Leu Ala Val Gly LeuAla Val Phe Phe Phe 20 25 30 8 14 PRT Homo sapiens 8 Arg Glu Val Pro TyrAla Tyr Ile Arg Glu Gly His Glu Lys 1 5 10 9 14 PRT Homo sapiens 9 AspGly Asn Phe Asp Thr Val Ala Met Leu Arg Gly Glu Met 1 5 10 10 15 PRTHomo sapiens 10 Pro Lys Ser Ala Leu Arg Asp Trp Met Gly Cys Pro Ser GlyGly 1 5 10 15 11 489 PRT Unknown X = UNKNOWN 11 Met Ala Pro Ala Ala TrpLeu Arg Ser Ala Ala Ala Arg Ala Leu Leu 1 5 10 15 Pro Pro Met Leu LeuLeu Leu Leu Gln Pro Pro Pro Leu Leu Ala Arg 20 25 30 Ala Leu Pro Pro AspVal His His Leu His Ala Glu Arg Arg Gly Pro 35 40 45 Gln Pro Trp His AlaAla Leu Pro Ser Ser Pro Ala Pro Ala Pro Ala 50 55 60 Thr Gln Glu Ala ProArg Pro Ala Ser Ser Leu Arg Pro Pro Arg Cys 65 70 75 80 Gly Val Pro AspPro Ser Asp Gly Leu Ser Ala Arg Asn Arg Gln Lys 85 90 95 Arg Phe Val LeuSer Gly Gly Arg Trp Glu Lys Thr Asp Leu Thr Tyr 100 105 110 Arg Ile LeuArg Phe Pro Trp Gln Leu Val Gln Glu Gln Val Arg Gln 115 120 125 Thr MetAla Glu Ala Leu Lys Val Trp Ser Asp Val Thr Pro Leu Thr 130 135 140 PheThr Glu Val His Glu Gly Arg Ala Asp Ile Met Ile Asp Phe Ala 145 150 155160 Arg Tyr Trp Asp Gly Asp Asp Leu Pro Phe Asp Gly Pro Gly Gly Ile 165170 175 Leu Ala His Ala Phe Phe Pro Lys Thr His Arg Glu Gly Asp Val His180 185 190 Phe Asp Tyr Asp Glu Thr Trp Thr Ile Gly Asp Asp Gln Gly ThrAsp 195 200 205 Leu Leu Gln Val Ala Ala His Glu Phe Gly His Val Leu GlyLeu Gln 210 215 220 His Thr Thr Ala Ala Lys Ala Leu Met Ser Ala Phe TyrThr Phe Arg 225 230 235 240 Tyr Pro Leu Ser Leu Ser Pro Asp Asp Cys ArgGly Val Gln His Leu 245 250 255 Tyr Gly Gln Pro Trp Pro Thr Val Thr SerArg Thr Pro Ala Leu Gly 260 265 270 Pro Gln Ala Gly Ile Asp Thr Asn GluIle Ala Pro Leu Glu Pro Asp 275 280 285 Ala Pro Pro Asp Ala Cys Glu AlaSer Phe Asp Ala Val Ser Thr Ile 290 295 300 Arg Gly Glu Leu Phe Phe PheLys Ala Gly Phe Val Trp Arg Leu Arg 305 310 315 320 Gly Gly Gln Leu GlnPro Gly Tyr Pro Ala Leu Ala Ser Arg His Trp 325 330 335 Gln Gly Leu ProSer Pro Val Asp Ala Ala Phe Glu Asp Ala Gln Gly 340 345 350 His Ile TrpPhe Phe Gln Gly Ala Gln Tyr Trp Val Tyr Asp Gly Glu 355 360 365 Lys ProVal Leu Gly Pro Ala Pro Leu Thr Glu Leu Gly Leu Val Arg 370 375 380 PhePro Val His Ala Ala Leu Val Trp Gly Pro Glu Lys Asn Lys Ile 385 390 395400 Tyr Phe Phe Arg Gly Arg Asp Tyr Trp Arg Phe His Pro Ser Thr Arg 405410 415 Arg Val Asp Ser Pro Val Pro Arg Arg Ala Thr Asp Trp Arg Gly Val420 425 430 Pro Ser Glu Ile Asp Ala Ala Phe Gln Asp Ala Asp Gly Tyr AlaTyr 435 440 445 Phe Leu Arg Gly Arg Leu Tyr Trp Lys Phe Asp Pro Val LysVal Lys 450 455 460 Ala Leu Glu Gly Phe Pro Arg Leu Val Gly Pro Asp PhePhe Gly Cys 465 470 475 480 Ala Glu Pro Ala Asn Thr Phe Leu Xaa 485 12469 PRT Unknown Description of Unknown Organism Known Member of MatrixMetalloproteinase Family 12 Met His Ser Phe Pro Pro Leu Leu Leu Leu LeuPhe Trp Gly Val Val 1 5 10 15 Ser His Ser Phe Pro Ala Thr Leu Glu ThrGln Glu Gln Asp Val Asp 20 25 30 Leu Val Gln Lys Tyr Leu Glu Lys Tyr TyrAsn Leu Lys Asn Asp Gly 35 40 45 Arg Gln Val Glu Lys Arg Arg Asn Ser GlyPro Val Val Glu Lys Leu 50 55 60 Lys Gln Met Gln Glu Phe Phe Gly Leu LysVal Thr Gly Lys Pro Asp 65 70 75 80 Ala Glu Thr Leu Lys Val Met Lys GlnPro Arg Cys Gly Val Pro Asp 85 90 95 Val Ala Gln Phe Val Leu Thr Glu GlyAsn Pro Arg Trp Glu Gln Thr 100 105 110 His Leu Thr Tyr Arg Ile Glu AsnTyr Thr Pro Asp Leu Pro Arg Ala 115 120 125 Asp Val Asp His Ala Ile GluLys Ala Phe Gln Leu Trp Ser Asn Val 130 135 140 Thr Pro Leu Thr Phe ThrLys Val Ser Glu Gly Gln Ala Asp Ile Met 145 150 155 160 Ile Ser Phe ValArg Gly Asp His Arg Asp Asn Ser Pro Phe Asp Gly 165 170 175 Pro Gly GlyAsn Leu Ala His Ala Phe Gln Pro Gly Pro Gly Ile Gly 180 185 190 Gly AspAla His Phe Asp Glu Asp Glu Arg Trp Thr Asn Asn Phe Thr 195 200 205 GluTyr Asn Leu His Arg Val Ala Ala His Glu Leu Gly His Ser Leu 210 215 220Gly Leu Ser His Ser Thr Asp Ile Gly Ala Leu Met Tyr Pro Ser Tyr 225 230235 240 Thr Phe Ser Gly Asp Val Gln Leu Ala Gln Asp Asp Ile Asp Gly Ile245 250 255 Gln Ala Ile Tyr Gly Arg Ser Gln Asn Pro Val Gln Pro Ile GlyPro 260 265 270 Gln Thr Pro Lys Ala Cys Asp Ser Lys Leu Thr Phe Asp AlaIle Thr 275 280 285 Thr Ile Arg Gly Glu Val Met Phe Phe Lys Asp Arg PheTyr Met Arg 290 295 300 Thr Asn Pro Phe Tyr Pro Glu Val Glu Leu Asn PheThr Ser Val Phe 305 310 315 320 Trp Pro Gln Leu Pro Asn Gly Leu Glu AlaAla Tyr Glu Phe Ala Asp 325 330 335 Arg Asp Glu Val Arg Phe Phe Lys GlyAsn Lys Tyr Trp Ala Val Gln 340 345 350 Gly Gln Asn Val Leu His Gly TyrPro Lys Asp Ile Tyr Ser Ser Phe 355 360 365 Gly Phe Pro Arg Thr Val LysHis Ile Asp Ala Ala Leu Ser Glu Glu 370 375 380 Asn Thr Gly Lys Thr TyrPhe Phe Val Ala Asn Lys Tyr Trp Arg Tyr 385 390 395 400 Asp Glu Tyr LysArg Ser Met Asp Pro Gly Tyr Pro Lys Met Ile Ala 405 410 415 His Asp PhePro Gly Ile Gly His Lys Val Asp Ala Val Phe Met Lys 420 425 430 Asp GlyPhe Phe Tyr Phe Phe His Gly Thr Arg Gln Tyr Lys Phe Asp 435 440 445 ProLys Thr Lys Arg Ile Leu Thr Leu Gln Lys Ala Asn Ser Trp Phe 450 455 460Asn Cys Arg Lys Asn 465 13 468 PRT Unknown X = UNKNOWN 13 Met Phe SerLeu Lys Thr Leu Pro Phe Leu Leu Leu Leu His Val Gln 1 5 10 15 Ile SerLys Ala Phe Pro Val Ser Ser Lys Glu Lys Asn Thr Lys Thr 20 25 30 Val GlnAsp Tyr Leu Glu Lys Phe Tyr Gln Leu Pro Ser Asn Gln Tyr 35 40 45 Gln SerThr Arg Lys Asn Gly Thr Asn Val Ile Val Glu Lys Leu Lys 50 55 60 Glu MetGln Arg Phe Phe Gly Leu Asn Val Thr Gly Lys Pro Asn Glu 65 70 75 80 GluThr Leu Asp Met Met Lys Lys Pro Arg Cys Gly Val Pro Asp Ser 85 90 95 GlyGly Phe Met Leu Thr Pro Gly Asn Pro Lys Trp Glu Arg Thr Asn 100 105 110Leu Thr Tyr Arg Ile Arg Asn Tyr Thr Pro Gln Leu Ser Glu Ala Glu 115 120125 Val Glu Arg Ala Ile Lys Asp Ala Phe Glu Leu Trp Ser Val Ala Ser 130135 140 Pro Leu Ile Phe Thr Arg Ile Ser Gln Gly Glu Ala Asp Ile Asn Ile145 150 155 160 Ala Phe Tyr Gln Arg Asp His Gly Asp Asn Ser Pro Phe AspGly Pro 165 170 175 Asn Gly Ile Leu Ala His Ala Phe Gln Pro Gly Gln GlyIle Gly Gly 180 185 190 Asp Ala His Phe Asp Ala Glu Glu Thr Trp Thr AsnThr Ser Ala Asn 195 200 205 Tyr Asn Leu Phe Leu Val Ala Ala His Glu PheGly His Ser Leu Gly 210 215 220 Leu Ala His Ser Ser Asp Pro Gly Ala LeuMet Tyr Pro Asn Tyr Ala 225 230 235 240 Phe Arg Glu Thr Ser Asn Tyr SerLeu Pro Gln Asp Asp Ile Asp Gly 245 250 255 Ile Gln Ala Ile Tyr Gly LeuSer Ser Asn Pro Ile Gln Pro Thr Gly 260 265 270 Pro Ser Thr Pro Lys ProCys Asp Pro Ser Leu Thr Phe Asp Ala Ile 275 280 285 Thr Thr Leu Arg GlyGlu Ile Leu Phe Phe Lys Asp Arg Tyr Phe Trp 290 295 300 Arg Arg His ProGln Leu Gln Arg Val Glu Met Asn Phe Ile Ser Leu 305 310 315 320 Phe TrpPro Ser Leu Pro Thr Gly Ile Gln Ala Ala Tyr Glu Asp Phe 325 330 335 AspArg Asp Leu Ile Phe Leu Phe Lys Gly Asn Gln Tyr Trp Ala Leu 340 345 350Ser Gly Tyr Asp Ile Leu Gln Gly Tyr Pro Lys Asp Ile Ser Asn Tyr 355 360365 Gly Phe Pro Ser Ser Val Gln Ala Ile Asp Ala Ala Val Phe Tyr Arg 370375 380 Ser Lys Thr Tyr Phe Phe Val Asn Asp Gln Phe Trp Arg Tyr Asp Asn385 390 395 400 Gln Arg Gln Phe Met Glu Pro Gly Tyr Pro Lys Ser Ile SerGly Ala 405 410 415 Phe Pro Gly Ile Glu Ser Lys Val Asp Ala Val Phe GlnGln Glu His 420 425 430 Phe Phe His Val Phe Ser Gly Pro Arg Tyr Tyr AlaPhe Asp Leu Ile 435 440 445 Ala Gln Arg Val Thr Arg Val Ala Arg Gly AsnLys Trp Leu Asn Cys 450 455 460 Arg Tyr Gly Xaa 465 14 476 PRT UnknownDescription of Unknown Organism Known Member of Matrix MetalloproteinaseFamily 14 Met Met His Leu Ala Phe Leu Val Leu Leu Cys Leu Pro Val CysSer 1 5 10 15 Ala Tyr Pro Leu Ser Gly Ala Ala Lys Glu Glu Asp Ser AsnLys Asp 20 25 30 Leu Ala Gln Gln Tyr Leu Glu Lys Tyr Tyr Asn Leu Glu LysAsp Val 35 40 45 Lys Gln Phe Arg Arg Lys Asp Ser Asn Leu Ile Val Lys LysIle Gln 50 55 60 Gly Met Gln Lys Phe Leu Gly Leu Glu Val Thr Gly Lys LeuAsp Thr 65 70 75 80 Asp Thr Leu Glu Val Met Arg Lys Pro Arg Cys Gly ValPro Asp Val 85 90 95 Gly His Phe Ser Ser Phe Pro Gly Met Pro Lys Trp ArgLys Thr His 100 105 110 Leu Thr Tyr Arg Ile Val Asn Tyr Thr Pro Asp LeuPro Arg Asp Ala 115 120 125 Val Asp Ser Ala Ile Glu Lys Ala Leu Lys ValTrp Glu Glu Val Thr 130 135 140 Pro Leu Thr Phe Ser Arg Leu Tyr Glu GlyGlu Ala Asp Ile Met Ile 145 150 155 160 Ser Phe Ala Val Lys Glu His GlyAsp Phe Tyr Ser Phe Asp Gly Pro 165 170 175 Gly His Ser Leu Ala His AlaTyr Pro Pro Gly Pro Gly Leu Tyr Gly 180 185 190 Asp Ile His Phe Asp AspAsp Glu Lys Trp Thr Glu Asp Ala Ser Gly 195 200 205 Thr Asn Leu Phe LeuVal Ala Ala His Glu Leu Gly His Ser Leu Gly 210 215 220 Leu Phe His SerAla Asn Thr Glu Ala Leu Met Tyr Pro Leu Tyr Asn 225 230 235 240 Ser PheThr Glu Leu Ala Gln Phe Arg Leu Ser Gln Asp Asp Val Asn 245 250 255 GlyIle Gln Ser Leu Tyr Gly Pro Pro Pro Ala Ser Thr Glu Glu Pro 260 265 270Leu Val Pro Thr Lys Ser Val Pro Ser Gly Ser Glu Met Pro Ala Lys 275 280285 Cys Asp Pro Ala Leu Ser Phe Asp Ala Ile Ser Thr Leu Arg Gly Glu 290295 300 Tyr Leu Phe Phe Lys Asp Arg Tyr Phe Trp Arg Arg Ser His Trp Asn305 310 315 320 Pro Glu Pro Glu Phe His Leu Ile Ser Ala Phe Trp Pro SerLeu Pro 325 330 335 Ser Tyr Leu Asp Ala Ala Tyr Glu Val Asn Ser Arg AspThr Val Phe 340 345 350 Ile Phe Lys Gly Asn Glu Phe Trp Ala Ile Arg GlyAsn Glu Val Gln 355 360 365 Ala Gly Tyr Pro Arg Gly Ile His Thr Leu GlyPhe Pro Pro Thr Ile 370 375 380 Arg Lys Ile Asp Ala Ala Val Ser Asp LysGlu Lys Lys Lys Thr Tyr 385 390 395 400 Phe Phe Ala Ala Asp Lys Tyr TrpArg Phe Asp Glu Asn Ser Gln Ser 405 410 415 Met Glu Gln Gly Phe Pro ArgLeu Ile Ala Asp Asp Phe Pro Gly Val 420 425 430 Glu Pro Lys Val Asp AlaVal Leu Gln Ala Phe Gly Phe Phe Tyr Phe 435 440 445 Phe Ser Gly Ser SerGln Phe Glu Phe Asp Pro Asn Ala Arg Met Val 450 455 460 Thr His Ile LeuLys Ser Asn Ser Trp Leu His Cys 465 470 475 15 477 PRT UnknownDescription of Unknown Organism Known Member of Matrix MetalloproteinaseFamily 15 Met Lys Ser Leu Pro Ile Leu Leu Leu Leu Cys Val Ala Val CysSer 1 5 10 15 Ala Tyr Pro Leu Asp Gly Ala Ala Arg Gly Glu Asp Thr SerMet Asn 20 25 30 Leu Val Gln Lys Tyr Leu Glu Asn Tyr Tyr Asp Leu Lys LysAsp Val 35 40 45 Lys Gln Phe Val Arg Arg Lys Asp Ser Gly Pro Val Val LysLys Ile 50 55 60 Arg Glu Met Gln Lys Phe Leu Gly Leu Glu Val Thr Gly LysLeu Asp 65 70 75 80 Ser Asp Thr Leu Glu Val Met Arg Lys Pro Arg Cys GlyVal Pro Asp 85 90 95 Val Gly His Phe Arg Thr Phe Pro Gly Ile Pro Lys TrpArg Lys Thr 100 105 110 His Leu Thr Tyr Arg Ile Val Asn Tyr Thr Pro AspLeu Pro Lys Asp 115 120 125 Ala Val Asp Ser Ala Val Glu Lys Ala Leu LysVal Trp Glu Glu Val 130 135 140 Thr Pro Leu Thr Phe Ser Arg Leu Tyr GluGly Glu Ala Asp Ile Met 145 150 155 160 Ile Ser Phe Ala Val Arg Glu HisGly Asp Phe Tyr Pro Phe Asp Gly 165 170 175 Pro Gly Asn Val Leu Ala HisAla Tyr Ala Pro Gly Pro Gly Ile Asn 180 185 190 Gly Asp Ala His Phe AspAsp Asp Glu Gln Trp Thr Lys Asp Thr Thr 195 200 205 Gly Thr Asn Leu PheLeu Val Ala Ala His Glu Ile Gly His Ser Leu 210 215 220 Gly Leu Phe HisSer Ala Asn Thr Glu Ala Leu Met Tyr Pro Leu Tyr 225 230 235 240 His SerLeu Thr Asp Leu Thr Arg Phe Arg Leu Ser Gln Asp Asp Ile 245 250 255 AsnGly Ile Gln Ser Leu Tyr Gly Pro Pro Pro Asp Ser Pro Glu Thr 260 265 270Pro Leu Val Pro Thr Glu Pro Val Pro Pro Glu Pro Gly Thr Pro Ala 275 280285 Asn Cys Asp Pro Ala Leu Ser Phe Asp Ala Val Ser Thr Leu Arg Gly 290295 300 Glu Ile Leu Ile Phe Lys Asp Arg His Phe Trp Arg Lys Ser Leu Arg305 310 315 320 Lys Leu Glu Pro Glu Leu His Leu Ile Ser Ser Phe Trp ProSer Leu 325 330 335 Pro Ser Gly Val Asp Ala Ala Tyr Glu Val Thr Ser LysAsp Leu Val 340 345 350 Phe Ile Phe Lys Gly Asn Gln Phe Trp Ala Ile ArgGly Asn Glu Val 355 360 365 Arg Ala Gly Tyr Pro Arg Gly Ile His Thr LeuGly Phe Pro Pro Thr 370 375 380 Val Arg Lys Ile Asp Ala Ala Ile Ser AspLys Glu Lys Asn Lys Thr 385 390 395 400 Tyr Phe Phe Val Glu Asp Lys TyrTrp Arg Phe Asp Glu Lys Arg Asn 405 410 415 Ser Met Glu Pro Gly Phe ProLys Gln Ile Ala Glu Asp Phe Pro Gly 420 425 430 Ile Asp Ser Lys Ile AspAla Val Phe Glu Glu Phe Gly Phe Phe Tyr 435 440 445 Phe Phe Thr Gly SerSer Gln Leu Glu Phe Asp Pro Asn Ala Lys Lys 450 455 460 Val Thr His ThrLeu Lys Ser Asn Ser Trp Leu Asn Cys 465 470 475 16 708 PRT Unknown X =UNKNOWN 16 Met Ser Leu Trp Gln Pro Leu Val Leu Val Leu Leu Val Leu GlyCys 1 5 10 15 Cys Phe Ala Ala Pro Arg Gln Arg Gln Ser Thr Leu Val LeuPhe Pro 20 25 30 Gly Asp Leu Arg Thr Asn Leu Thr Asp Arg Gln Leu Ala GluGlu Tyr 35 40 45 Leu Tyr Arg Tyr Gly Tyr Thr Arg Val Ala Glu Met Arg GlyGlu Ser 50 55 60 Lys Ser Leu Gly Pro Ala Leu Leu Leu Leu Gln Lys Gln LeuSer Leu 65 70 75 80 Pro Glu Thr Gly Glu Leu Asp Ser Ala Thr Leu Lys AlaMet Arg Thr 85 90 95 Pro Arg Cys Gly Val Pro Asp Leu Gly Arg Phe Gln ThrPhe Glu Gly 100 105 110 Asp Leu Lys Trp His His His Asn Ile Thr Tyr TrpIle Gln Asn Tyr 115 120 125 Ser Glu Asp Leu Pro Arg Ala Val Ile Asp AspAla Phe Ala Arg Ala 130 135 140 Phe Ala Leu Trp Ser Ala Val Thr Pro LeuThr Phe Thr Arg Val Tyr 145 150 155 160 Ser Arg Asp Ala Asp Ile Val IleGln Phe Gly Val Ala Glu His Gly 165 170 175 Asp Gly Tyr Pro Phe Asp GlyLys Asp Gly Leu Leu Ala His Ala Phe 180 185 190 Pro Pro Gly Pro Gly IleGln Gly Asp Ala His Phe Asp Asp Asp Glu 195 200 205 Leu Trp Ser Leu GlyLys Gly Val Val Val Pro Thr Arg Phe Gly Asn 210 215 220 Ala Asp Gly AlaAla Cys His Phe Pro Phe Ile Phe Glu Gly Arg Ser 225 230 235 240 Tyr SerAla Cys Thr Thr Asp Gly Arg Ser Asp Gly Leu Pro Trp Cys 245 250 255 SerThr Thr Ala Asn Tyr Asp Thr Asp Asp Arg Phe Gly Phe Cys Pro 260 265 270Ser Glu Arg Leu Tyr Thr Arg Asp Gly Asn Ala Asp Gly Lys Pro Cys 275 280285 Gln Phe Pro Phe Ile Phe Gln Gly Gln Ser Tyr Ser Ala Cys Thr Thr 290295 300 Asp Gly Arg Ser Asp Gly Tyr Arg Trp Cys Ala Thr Thr Ala Asn Tyr305 310 315 320 Asp Arg Asp Lys Leu Phe Gly Phe Cys Pro Thr Arg Ala AspSer Thr 325 330 335 Val Met Gly Gly Asn Ser Ala Gly Glu Leu Cys Val PhePro Phe Thr 340 345 350 Phe Leu Gly Lys Glu Tyr Ser Thr Cys Thr Ser GluGly Arg Gly Asp 355 360 365 Gly Arg Leu Trp Cys Ala Thr Thr Ser Asn PheAsp Ser Asp Lys Lys 370 375 380 Trp Gly Phe Cys Pro Asp Gln Gly Tyr SerLeu Phe Leu Val Ala Ala 385 390 395 400 His Glu Phe Gly His Ala Leu GlyLeu Asp His Ser Ser Val Pro Glu 405 410 415 Ala Leu Met Tyr Pro Met TyrArg Phe Thr Glu Gly Pro Pro Leu His 420 425 430 Lys Asp Asp Val Asn GlyIle Arg His Leu Tyr Gly Pro Arg Pro Glu 435 440 445 Pro Glu Pro Arg ProPro Thr Thr Thr Thr Pro Gln Pro Thr Ala Pro 450 455 460 Pro Thr Val CysPro Thr Gly Pro Pro Thr Val His Pro Ser Glu Arg 465 470 475 480 Pro ThrAla Gly Pro Thr Gly Pro Pro Ser Ala Gly Pro Thr Gly Pro 485 490 495 ProThr Ala Gly Pro Ser Thr Ala Thr Thr Val Pro Leu Ser Pro Val 500 505 510Asp Asp Ala Cys Asn Val Asn Ile Phe Asp Ala Ile Ala Glu Ile Gly 515 520525 Asn Gln Leu Tyr Leu Phe Lys Asp Gly Lys Tyr Trp Arg Phe Ser Glu 530535 540 Gly Arg Gly Ser Arg Pro Gln Gly Pro Phe Leu Ile Ala Asp Lys Trp545 550 555 560 Pro Ala Leu Pro Arg Lys Leu Asp Ser Val Phe Glu Glu ProLeu Ser 565 570 575 Lys Lys Leu Phe Phe Phe Ser Gly Arg Gln Val Trp ValTyr Thr Gly 580 585 590 Ala Ser Val Leu Gly Pro Arg Arg Leu Asp Lys LeuGly Leu Gly Ala 595 600 605 Asp Val Ala Gln Val Thr Gly Ala Leu Arg SerGly Arg Gly Lys Met 610 615 620 Leu Leu Phe Ser Gly Arg Arg Leu Trp ArgPhe Asp Val Lys Ala Gln 625 630 635 640 Met Val Asp Pro Arg Ser Ala SerGlu Val Asp Arg Met Phe Pro Gly 645 650 655 Val Pro Leu Asp Thr His AspVal Phe Gln Tyr Arg Glu Lys Ala Tyr 660 665 670 Phe Cys Gln Asp Arg PheTyr Trp Arg Val Ser Ser Arg Ser Glu Leu 675 680 685 Asn Gln Val Asp GlnVal Gly Tyr Val Thr Tyr Asp Ile Leu Gln Cys 690 695 700 Pro Glu Asp Xaa705 17 631 PRT Unknown Description of Unknown Organism Known Member ofMatrix Metalloproteinase Family 17 Ala Pro Ser Pro Ile Ile Lys Phe ProGly Asp Val Ala Pro Lys Thr 1 5 10 15 Asp Lys Glu Leu Ala Val Gln TyrLeu Asn Thr Phe Tyr Gly Cys Pro 20 25 30 Lys Glu Ser Cys Asn Leu Phe ValLeu Lys Asp Thr Leu Lys Lys Met 35 40 45 Gln Lys Phe Phe Gly Leu Pro GlnThr Gly Asp Leu Asp Gln Asn Thr 50 55 60 Ile Glu Thr Met Arg Lys Pro ArgCys Gly Asn Pro Asp Val Ala Asn 65 70 75 80 Tyr Asn Phe Phe Pro Arg LysPro Lys Trp Asp Lys Asn Gln Ile Thr 85 90 95 Tyr Arg Ile Ile Gly Tyr ThrPro Asp Leu Asp Pro Glu Thr Val Asp 100 105 110 Asp Ala Phe Ala Arg AlaPhe Gln Val Trp Ser Asp Val Thr Pro Leu 115 120 125 Arg Phe Ser Arg IleHis Asp Gly Glu Ala Asp Ile Met Ile Asn Phe 130 135 140 Gly Arg Trp GluHis Gly Asp Gly Tyr Pro Phe Asp Gly Lys Asp Gly 145 150 155 160 Leu LeuAla His Ala Phe Ala Pro Gly Thr Gly Val Gly Gly Asp Ser 165 170 175 HisPhe Asp Asp Asp Glu Leu Trp Thr Leu Gly Glu Gly Gln Val Val 180 185 190Arg Val Lys Tyr Gly Asn Ala Asp Gly Glu Tyr Cys Lys Phe Pro Phe 195 200205 Leu Phe Asn Gly Lys Glu Tyr Asn Ser Cys Thr Asp Thr Gly Arg Ser 210215 220 Asp Gly Phe Leu Trp Cys Ser Thr Thr Tyr Asn Phe Glu Lys Asp Gly225 230 235 240 Lys Tyr Gly Phe Cys Pro His Glu Ala Leu Phe Thr Met GlyGly Asn 245 250 255 Ala Glu Gly Gln Pro Cys Lys Phe Pro Phe Arg Phe GlnGly Thr Ser 260 265 270 Tyr Asp Ser Cys Thr Thr Glu Gly Arg Thr Asp GlyTyr Arg Trp Cys 275 280 285 Gly Thr Thr Glu Asp Tyr Asp Arg Asp Lys LysTyr Gly Phe Cys Pro 290 295 300 Glu Thr Ala Met Ser Thr Val Gly Gly AsnSer Glu Gly Ala Pro Cys 305 310 315 320 Val Phe Pro Phe Thr Phe Leu GlyAsn Lys Tyr Glu Ser Cys Thr Ser 325 330 335 Ala Gly Arg Ser Asp Gly LysMet Trp Cys Ala Thr Thr Ala Asn Tyr 340 345 350 Asp Asp Asp Arg Lys TrpGly Phe Cys Pro Asp Gln Gly Tyr Ser Leu 355 360 365 Phe Leu Val Ala AlaHis Glu Phe Gly His Ala Met Gly Leu Glu His 370 375 380 Ser Gln Asp ProGly Ala Leu Met Ala Pro Ile Tyr Thr Tyr Thr Lys 385 390 395 400 Asn PheArg Leu Ser Gln Asp Asp Ile Lys Gly Ile Gln Glu Leu Tyr 405 410 415 GlyAla Ser Pro Asp Ile Asp Leu Gly Thr Gly Pro Thr Pro Thr Leu 420 425 430Gly Pro Val Thr Pro Glu Ile Cys Lys Gln Asp Ile Val Phe Asp Gly 435 440445 Ile Ala Gln Ile Arg Gly Glu Ile Phe Phe Phe Lys Asp Arg Phe Ile 450455 460 Trp Arg Thr Val Thr Pro Arg Asp Lys Pro Met Gly Pro Leu Leu Val465 470 475 480 Ala Thr Phe Trp Pro Glu Leu Pro Glu Lys Ile Asp Ala ValTyr Glu 485 490 495 Ala Pro Gln Glu Glu Lys Ala Val Phe Phe Ala Gly AsnGlu Tyr Trp 500 505 510 Ile Tyr Ser Ala Ser Thr Leu Glu Arg Gly Tyr ProLys Pro Leu Thr 515 520 525 Ser Leu Gly Leu Pro Pro Asp Val Gln Arg ValAsp Ala Ala Phe Asn 530 535 540 Trp Ser Lys Asn Lys Lys Thr Tyr Ile PheAla Gly Asp Lys Phe Trp 545 550 555 560 Arg Tyr Asn Glu Val Lys Lys LysMet Asp Pro Gly Phe Pro Lys Leu 565 570 575 Ile Ala Asp Ala Trp Asn AlaIle Pro Asp Asn Leu Asp Ala Val Val 580 585 590 Asp Leu Gln Gly Gly GlyHis Ser Tyr Phe Phe Lys Gly Ala Tyr Tyr 595 600 605 Leu Lys Leu Glu AsnGln Ser Leu Lys Ser Val Lys Phe Gly Ser Ile 610 615 620 Lys Ser Asp TrpLeu Gly Cys 625 630 18 267 PRT Unknown Description of Unknown OrganismKnown Member of Matrix Metalloproteinase Family 18 Met Arg Leu Thr ValLeu Cys Ala Val Cys Leu Leu Pro Gly Ser Leu 1 5 10 15 Ala Leu Pro LeuPro Gln Glu Ala Gly Gly Met Ser Glu Leu Gln Trp 20 25 30 Glu Gln Ala GlnAsp Tyr Leu Lys Arg Phe Tyr Leu Tyr Asp Ser Glu 35 40 45 Thr Lys Asn AlaAsn Ser Leu Glu Ala Lys Leu Lys Glu Met Gln Lys 50 55 60 Phe Phe Gly LeuPro Ile Thr Gly Met Leu Asn Ser Arg Val Ile Glu 65 70 75 80 Ile Met GlnLys Pro Arg Cys Gly Val Pro Asp Val Ala Glu Tyr Ser 85 90 95 Leu Phe ProAsn Ser Pro Lys Trp Thr Ser Lys Val Val Thr Tyr Arg 100 105 110 Ile ValSer Tyr Thr Arg Asp Leu Pro His Ile Thr Val Asp Arg Leu 115 120 125 ValSer Lys Ala Leu Asn Met Trp Gly Lys Glu Ile Pro Leu His Phe 130 135 140Arg Lys Val Val Trp Gly Thr Ala Asp Ile Met Ile Gly Phe Ala Arg 145 150155 160 Gly Ala His Gly Asp Ser Tyr Pro Phe Asp Gly Pro Gly Asn Thr Leu165 170 175 Ala His Ala Phe Ala Pro Gly Thr Gly Leu Gly Gly Asp Ala HisPhe 180 185 190 Asp Glu Asp Glu Arg Trp Thr Asp Gly Ser Ser Leu Gly IleAsn Phe 195 200 205 Leu Tyr Ala Ala Thr His Glu Leu Gly His Ser Leu GlyMet Gly His 210 215 220 Ser Ser Asp Pro Asn Ala Val Met Tyr Pro Thr TyrGly Asn Gly Asp 225 230 235 240 Pro Gln Asn Phe Lys Leu Ser Gln Asp AspIle Lys Gly Ile Gln Lys 245 250 255 Leu Tyr Gly Lys Arg Ser Asn Ser ArgLys Lys 260 265 19 231 PRT Unknown Description of Unknown Organism KnownMember of Matrix Metalloproteinase Family 19 Met Pro Leu Leu Leu Leu LeuGlu Tyr Leu Glu Lys Leu Met Gln Lys 1 5 10 15 Phe Gly Leu Val Thr GlyLys Leu Asp Thr Leu Met Arg Lys Pro Arg 20 25 30 Cys Gly Val Pro Asp ValGly Phe Phe Pro Gly Pro Lys Trp Thr Leu 35 40 45 Thr Tyr Arg Ile Asn TyrThr Pro Asp Leu Pro Val Asp Ala Lys Ala 50 55 60 Phe Val Trp Ser Val ThrPro Leu Thr Phe Arg Val Glu Gly Ala Asp 65 70 75 80 Ile Met Ile Phe AlaHis Gly Asp Tyr Pro Phe Asp Gly Pro Gly Gly 85 90 95 Leu Ala His Ala PhePro Gly Pro Gly Ile Gly Gly Asp Ala His Phe 100 105 110 Asp Asp Glu TrpThr Asn Leu Phe Leu Val Ala Ala His Glu Gly His 115 120 125 Ser Leu GlyLeu His Ser Asp Pro Ala Leu Met Tyr Pro Thr Phe Phe 130 135 140 Leu SerGln Asp Asp Ile Gly Ile Gln Leu Tyr Gly Pro Pro Thr Cys 145 150 155 160Asp Phe Asp Ala Ile Thr Arg Gly Glu Phe Phe Lys Asp Arg Trp Arg 165 170175 Leu Ser Phe Trp Pro Leu Pro Asp Ala Ala Tyr Glu Phe Phe Gly Asn 180185 190 Tyr Trp Gly Gly Tyr Pro Ile Leu Gly Pro Val Asp Ala Ala Lys Thr195 200 205 Tyr Phe Phe Lys Trp Arg Asp Met Pro Gly Pro Ile Phe Pro GlyAsp 210 215 220 Ala Val Phe Phe Trp Leu Cys 225 230

What is claimed is:
 1. An isolated native membrane-typematrix-metalloproteinase, comprising an amino acid sequence from aminoacid numbers 160 to 173, 320 to 333, 498 to 512, and a continuoussequence of hydrophobic amino acids peculiar to membrane-bindingproteins from amino acid number 533 to 562 in the C terminus domainshown in SEQ ID NO:1.
 2. An isolated native membrane-typematrix-metalloproteinase according to claim 1, having a length of 582amino acids.
 3. An isolated native membrane-typematrix-metalloproteinase comprising an amino acid sequence from aminoacid number 1 to 173, 320 to 333 and 498 to 512 and 563 to 582, and acontinuous sequence of hydrophobic amino acids peculiar tomembrane-binding proteins from amino acid number 533 to 562 in the Cterminus domain shown in SEQ ID NO:1.
 4. An isolated protein having theamino acid sequence shown in SEQ ID NO:1.
 5. An isolated nativemembrane-type matrix-metalloproteinase having a length of 582 aminoacids and encoded by the nucleotide sequence of SEQ ID NO:2 or a DNAwhich hybridizes thereto under stringent conditions, wherein saidhybridization is carried out at 42° C. in the presence of 50% formamidewith washing at 68° C. in the presence of 0.15 M NaCl.